cells

Review Moonlighting in : Analysis of the Mitotic Functions of Transcription and Splicing Factors

1, 2,3, 2,4 1 Maria Patrizia Somma †, Evgeniya N. Andreyeva †, Gera A. Pavlova , Claudia Pellacani , Elisabetta Bucciarelli 1 , Julia V. Popova 2, Silvia Bonaccorsi 5 , Alexey V. Pindyurin 2,* and Maurizio Gatti 1,5,*

1 IBPM CNR c/o Department of Biology and Biotechnology, Sapienza University of Rome, 00185 Rome, Italy; [email protected] (M.P.S.); [email protected] (C.P.); [email protected] (E.B.) 2 Institute of Molecular and Cellular Biology, Siberian Branch of RAS, 630090 Novosibirsk, Russia; [email protected] (E.N.A.); [email protected] (G.A.P.); [email protected] (J.V.P.) 3 Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York, NY 10461, USA 4 Wellcome Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3BF, UK 5 Department of Biology and Biotechnology, Sapienza University of Rome, 00185 Rome, Italy; [email protected] * Correspondence: [email protected] (A.V.P.); [email protected] (M.G.) These authors contributed equally. †  Received: 9 May 2020; Accepted: 23 June 2020; Published: 26 June 2020 

Abstract: Moonlighting can perform one or more additional functions besides their primary role. It has been posited that a can acquire a moonlighting function through a gradual evolutionary process, which is favored when the primary and secondary functions are exerted in different cellular compartments. Transcription factors (TFs) and splicing factors (SFs) control processes that occur in interphase nuclei and are strongly reduced during , and are therefore in a favorable situation to evolve moonlighting mitotic functions. However, recently published moonlighting protein databases, which comprise almost 400 proteins, do not include TFs and SFs with secondary mitotic functions. We searched the literature and found several TFs and SFs with bona fide moonlighting mitotic functions, namely they localize to specific mitotic structure(s), interact with proteins enriched in the same structure(s), and are required for proper morphology and functioning of the structure(s). In addition, we describe TFs and SFs that localize to mitotic structures but cannot be classified as moonlighting proteins due to insufficient data on their biochemical interactions and mitotic roles. Nevertheless, we hypothesize that most TFs and SFs with specific mitotic localizations have either minor or redundant moonlighting functions, or are evolving towards the acquisition of these functions.

Keywords: transcription factors; splicing factors; multifunctional proteins; protein moonlighting; mitosis; microtubules; spindle; ; kinetochores; midbody

1. Introduction In 1999, Constance J. Jeffrey, coined the term “moonlighting protein” to describe a protein that performs one or more additional functions besides its canonical one [1]. Moonlighting proteins play roles in many different biological processes. Recently published manually curated databases (MoonProt 2.0 and MoonDB 2.0) describe almost 400 moonlighting proteins [2,3] and new bifunctional and multifunctional proteins are continuously discovered.

Cells 2020, 9, 1554; doi:10.3390/cells9061554 www.mdpi.com/journal/cells Cells 2020, 9, 1554 2 of 27

The coexistence of more than one activity in a single protein is the likely result of a gradual evolutionary transition from the original function to a novel function. It has been further posited that protein moonlighting is favored when the novel and the canonical function are exerted in different cellular compartments, and/or in combination with different binding partners, so as to avoid functional conflicts [1,4,5]. Under this view, mitosis seems to be an ideal process to favor acquisition of moonlighting functions, as it is characterized by a dramatic rearrangement of cell structures, accompanied by re-localization and compartmentalization of many proteins. For example, during condensation, many chromatin proteins are released in the cytoplasm and re-associate with the chromatin only when decondense during telophase. Some of these proteins have acquired moonlighting mitotic functions [6,7]. The ISWI and CHD4 chromatin remodeling factors bind the spindle microtubules (MTs) in both Drosophila S2 cells and Xenopus cells and egg extracts, and are required for spindle assembly and functioning [8,9]. Similarly, the INO80 ATPase, involved in both DNA replication and chromatin remodeling, binds the mitotic spindle and is required for chromosome segregation [10,11]. Thus, the mitotic spindle can bind and compartmentalize proteins that during interphase are associated with the chromatin and reside within the nucleus. During mitosis, several cellular processes such as transcription and splicing are strongly reduced [12–14], and therefore the proteins involved in these processes are in a very favorable situation to evolve secondary mitotic functions. Indeed, several genome-wide screens carried out in Drosophila and human cells have shown that RNAi-mediated depletion of many different transcription factors (TFs) and splicing factors (SFs) results in a variety of mitotic defects [15–20] (see Tables S1 and S2). Other TFs and SFs required for mitotic division have been identified in studies on specific proteins or protein complexes (see Tables1 and2 and text below). However, in most cases, it is unclear whether the TF- or SF-dependent mitotic is due to a direct (moonlighting) function of the factor, or to an indirect effect on transcription or splicing of one or more mRNAs encoding mitotic proteins (henceforth, mitotic mRNAs). In this review, we analyze the literature on the mitotic roles of TFs and SFs. We include in our TF category not only the canonical DNA binding factors, but also the proteins that regulate transcription without binding to DNA (e.g., co-activators and co-repressors) [21]. Similarly, we consider as SFs both the proteins complexed with small nuclear (snRNAs) and the proteins that do not directly interact with these RNAs but are required for splicing [22]. We report many examples of TFs and SFs that in addition to their canonical function in transcription and splicing, play moonlighting mitotic roles, as well as cases where the TFs and SFs appear to have indirect mitotic effects controlling the biogenesis of mitotic mRNAs. Defining whether a factor associated with a loss-of-function mitotic phenotype has a direct mitotic role is not straightforward. We classify a TF/SF as a moonlighting mitotic protein when it meets at least three conditions: (i) it localizes to a specific mitotic structure, (ii) biochemically interacts with proteins enriched in the same structure, and (iii) causes defects in this structure upon depletion. Conversely, a TF/SF is classified as an indirect mitotic player when it does not exhibit a mitotic localization and there is biochemical evidence that its loss impairs transcription or splicing of encoding mitotic proteins specifically related to the affected mitotic structure. However, for many TFs/SFs that localize to mitotic structures, published information is not sufficient to decide whether they play a direct function during cell division. For example, for some of these factors, there is no information on their biochemical interactions or loss-of-function mitotic phenotype, while knockdown of other factors does not appear to elicit a clear mitotic phenotype. Thus, we classify this heterogeneous group of TFs/SFs as factors with a “putatively direct” mitotic function (Tables1 and2), whose precise assessment requires further investigation. Overall, we provide a large but non-exhaustive list of TFs and SFs with mitotic functions and critically analyze their mitotic roles. We would like to point out that none of the TFs and SFs with direct mitotic roles (see Tables1 and2 below) are included in the extant moonlighting protein databases [ 2,3]. Cells 2020, 9, 1554 3 of 27

2. Transcription Factors (TFs) with Mitotic Functions

2.1. TFs That Control Mitosis by Regulating the Expression of Mitotic Genes Several TFs are thought to affect mitosis indirectly through the regulation of transcription or stability of mitotic mRNAs. Forkhead transcription factors (FKH-TFs) have been implicated in the regulation of mitotic transcription in both yeast and [23,24]. In humans, FKH-TFs (e.g., FOXO3) regulate the expression of mitotic genes such as cyclin B1 (CCNB1) and reductions in the activities of FKH-TFs result in delayed M-G1 transition and defective cytokinesis [23]. In S. pombe, most of the mitotic gene promoters targeted by FKH-TFs (e.g., Fkh2) are additionally associated with Sak1, a TF of the RFX family that also appears to control mitosis indirectly [24] (Table1). Another TF that is thought to have an indirect mitotic function is Zas1 of S. pombe, which controls the expression of a specific set of genes involved in cell division, including the gene encoding the Cnd1 subunit of the condensin complex [25]. Mutations in Zas1 cause defects in chromosome condensation and segregation, but these defects are not fully rescued by expression of the Cnd1 protein. This finding has led to the suggestion that the mitotic phenotype elicited by Zas1 deficiency might be the consequence of the simultaneous deregulation of multiple mitotic genes [25]. However, the observation that Zas1 remains associated with the chromosomes throughout mitosis, and that this association is essential for its mitotic function [25], does not exclude the possibility that Zas1 directly participates in the chromosome condensation process. A peculiar form of indirect mitotic regulation is exerted by the ERG of the ETS family. In addition to the nucleus, ERG accumulates in cytoplasmic messenger ribonucleoprotein (mRNP) aggregates called processing bodies (PBs), which contain the RNA degradation machinery. Consistent with this localization, ERG promotes mRNA decay by binding the CCR4–NOT deadenylation complex. ERG depletion in HeLa cells results in impaired degradation of a subset of mRNAs including those encoding the Aurora kinase A (AURKA) and Aurora kinase B (AURKB). Accumulation of these kinases deregulates Aurora signaling, resulting in a variety of mitotic defects, including fragmentation, morphologically abnormal bipolar spindles and multipolar spindles [26].

2.2. TFs That Localize to Centrosomes There are many TFs that accumulate at the centrosomes (Table1; Figure1). The centrosomes are self-assembling organelles that nucleate the spindle MTs during cell division. They are comprised of a pair of orthogonally arranged rod-like surrounded by (PCM), which contains several proteins necessary for MT nucleation, including γ- (TUBG1) [27,28]. In mammals, a pair consists of a mother centriole characterized by the presence of subdistal appendages, and a younger daughter centriole that grows from the mother during interphase; the two centrioles remain attached to each other until , when they are disengaged by separase [27]. Some TFs meet our three criteria for a moonlighting function at the centrosomes, namely they localize to the centrosomes, physically interact with centrosomal proteins and are required for centrosome structure, duplication, or MT nucleation activity. Other TFs meet only one or two of these three criteria, and it is therefore currently unclear whether they play direct functions at the centrosomes. CellsCells2020 2020, 9,, 9 1554, x FOR PEER REVIEW 4 of 428 of 27

Figure 1. Localization of transcription and splicing factors to mitotic structures of (top) and late telophase (bottom) cells. Data are from human (h), mouse (m), Drosophila (Dm), and S. pombe (Sp) cells and from spindles assembled in Xenopus egg extracts (Xe). Protein localization in the schematic metaphase is also observed in prometaphases and anaphases. Likewise, the protein localization pattern in the schematic late telophase is comparable to that seen in earlier stages of telophase. Of the proteins Cells 2020, 9, 1554 5 of 27

that localize to the centrosomes, most are present from to telophase, but some (depicted in green) are not detected in telophase centrosomes. For other centrosomal proteins (marked by an asterisk), lack of pertinent images did not permit an assessment of their presence in telophase centrosomes. The midbody at the center of the cellular bridge consists of a dark zone that contains interdigitating antiparallel MTs associated with several proteins, including centralspindlin (MKLP1/KIF23 and CYK4/RACGAP1), CIT-K, KIF14, and anillin. These and other proteins form a dense cluster (midbody ring) that impedes access of anti-tubulin antibodies, resulting in a dark zone after immunostaining. The flanking regions also contain several proteins, including those of the ESCRT machinery that mediates abscission, which can occur at either side of the dark zone. Localization of some proteins is + (P ), dephosphorylation (P−), or desumoylation (SUMO−) dependent. An example of a moonlighting TF with a centrosomal function is the well-known tumor suppressor BRCA1. BRCA1 is a multifunctional protein that is included in several different complexes primarily involved in DNA replication and repair. BRCA1 can also associate with the RNA polymerase II holoenzyme and some well-known TFs (TFIIF/GTF2F1, TFIIE/GTF2E1, and TFIIH/GTF2H1; here and henceforth, the commonly used abbreviation for a human protein and its HUGO-based symbol are separated by a slash), acting as a transcriptional regulator [29,30]. In addition, BRCA1 is enriched at the centrosomes where it interacts with the BRCA1-associated RING domain protein (BARD1) and the Obg-like ATPase 1 (OLA1) to form a complex with E3 activity that binds and ubiquitinates γ-tubulin [31–35]. BRCA1 downregulation causes centrosome amplification and fragmentation in cell lines from mammary tissues [36,37] and in mouse embryonic fibroblast cells [38]. The localization of BRCA1 at the centrosome, its biochemical interaction with γ-tubulin, and the centrosomal phenotype elicited by its deficiency strongly suggest that BRCA1 is directly involved in centrosome maintenance. The evolutionarily conserved BRCA1 protein is also required for spindle formation induced by sperm nuclei in cytoplasmic extracts from Xenopus laevis eggs. In this system [39], BRCA1 is slightly enriched at the spindle poles and physically interacts with BARD1 and the spindle-associated proteins TPX2, NuMa, and XRHAMM. BRCA1 immunodepletion results in morphologically aberrant spindles with unfocused poles and a higher MT density compared to spindles formed in BRCA1-nondepleted extracts [40]. Because Xenopus egg extracts can support mitosis independently of transcription and translation, requiring only synthesis of cyclin B [41,42], these findings strongly suggest that BRCA1 directly contributes to centrosome-independent spindle assembly in this in vitro system. Another remarkable example of a TF with a moonlighting role in centrosome assembly and function is ATF5, a member of the ATF/CREB family of TFs [43]. In human cells, ATF5 accumulates at the centrosomes from G1 through anaphase and dissociates from the centrosomes during late telophase. Consistent with its subcellular localization, ATF5 interacts with multiple centrosomal proteins, including γ-tubulin and pericentrin (PCNT), and is specifically enriched in a cylindrical structure that encircles the proximal end of the mother centriole [44]. ATF5 association with the centrosomes is regulated by SUMOylation; SUMOylated ATF5 does not localize to centrosomes and does not interact with centrosomal proteins [45]. RNAi-mediated downregulation of ATF5 results in impaired centrosomal accumulation of PCM components such as γ-tubulin and PCNT, centriolar fragmentation and formation of multipolar spindles [44]. Interestingly, during late telophase, ATF5 dissociates from centrosomes and accumulates in the midbody [44], the structure in the middle of the bridge that connects the daughter cells during completion of cytokinesis (Figure1). However, the ATF5 functional role at the midbody, if any, is currently unknown. The midbody forms when the overlapping plus ends of the central spindle MTs are compacted by cytokinetic ring constriction, so as to form a MT-dense region, called dark zone, associated with multiple proteins that impede staining with anti-tubulin antibodies [46,47]. The midbody has a defined protein composition and substructure [47,48], but here we will only refer to the dark zone (DZ) and its flanking regions (FR), because these are the only regions we can appreciate in published figures of late telophases stained for both tubulin and the TF or SF under study (Figure1). Cells 2020, 9, 1554 6 of 27

Another integral centriolar component is AKNA, an AT-hook TF that is required for neurogenesis in mouse brain [49]. Like ATF5, AKNA localizes to the mother centriole, where it is mostly enriched at the subdistal appendages. In contrast with the ATF5 behavior, AKNA dissociates from centrosomes during mitosis upon increased phosphorylation and re-associates with the centrosomes during late telophase/early G1. AKNA recruits and physically interacts with components of the γ-tubulin ring complex (γTuRC), and MT binding proteins such as EB1, P150-Glued (DCTN1), and . RNAi-mediated AKNA knockdown results in a significant reduction in centrosome-driven MT regrowth after MT depolymerization with nocodazole, but it is unclear whether this defect underlies a gross alteration in normal spindle assembly [49]. Additional TFs that are likely to play moonlighting functions at the centrosomes are the Y-box binding protein-1 (YB-1/YBX1) and OCT1/POU2F1. YB-1, which is overexpressed in many , has roles in transcription, translation, and splicing, localizes to the centrosomes of human cells in a phosphorylation-dependent manner, and physically interacts with γ-tubulin and PCNT. In YB-1-depleted cells, centrosomes are structurally abnormal and have reduced MT nucleation ability [50,51]. OCT1 is a well-known TF that regulates metabolism and tumorigenicity in humans and mice. Upon phosphorylation in its DNA binding domain, OCT1 localizes to the centrosomes, the central spindle and the midbody dark zone, and to a lesser extent to the kinetochores ([52] and references therein). OCT1 interacts with proteins that are also found in the centrosomes such as BRCA1 [53,54] and PARP-1 [55]. RNAi-mediated OCT1 depletion in HeLa cells results in abnormal mitosis. Similarly, immunodepletion of the OCT1 ortholog from Xenopus egg extracts causes defects in spindle assembly, a finding that provides further support for a moonlighting mitotic function of OCT1 [52]. The OCT1 Drosophila ortholog, Nubbin, does not appear to localize to centrosomes but is enriched at both the central spindle and the midbody flanking regions [56] (Figure1). It is currently unknown whether OCT1 and Nubbin have roles in cytokinesis [52,56]. There are several additional TFs that localize to the centrosomes, but it is currently unclear whether they play direct functions in these structures. One of these TFs is the intensively studied /TP53 protein that plays crucial roles in the maintenance of genome stability and tumor suppression [57]. p53 controls transcription of genes involved in a variety of cellular processes, including cell cycle, DNA repair, senescence, , and autophagy, but also has direct cytoplasmic roles in mitochondrial membrane permeabilization, apoptosis, and possibly autophagy inhibition [57]. p53 localizes to the centrosomes in a variety of human, chicken, and rodent cells [58–60]; in human cells, centrosomal localization of p53 is phosphorylation-dependent [61]. Loss of p53 results in centrosome amplification in both human and mouse cells [62–66]. Interestingly, recent work has shown that p53 depletion in human non-transformed cells results in centrosome fragmentation [67]. However, centrosome fragmentation was not observed in p53-deficient mouse cells and human cells, which instead showed centrosome amplification [67], as previously described [65]. Although it has been suggested that p53 might directly contribute to prevention of centrosome amplification [64,65] or fragmentation [67], a compelling evidence for a moonlighting role of p53 at the centrosome is still lacking. Other TFs that accumulate at the centrosomes of mammalian cells, but do not fit our three criteria for a moonlighting centrosomal function are SP1, SF-1/NR5A1, Kaiso/ZBTB33, CTCF, SNAP45/SNAPC2, and ELK1. SP1, which has been implicated in tumorigenesis, localizes to the centrosomes of mouse and human cells and its loss causes centrosome amplification and decreased centrosome-driven MT nucleation [68]. It has been suggested that SP1 deficiency results in the activation of TORC1 signaling, which leads to centrosome amplification [68]. SF-1 prevents centrosome overduplication in mouse cells; SF-1 depletion leads to an aberrant centrosomal accumulation and activation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which is thought to activate the AKT-dependent signaling cascade that leads to centrosome amplification [69,70]. Kaiso is a BTB/POZ zinc finger protein that acts as a transcriptional repressor; in human cells, it localizes to the centrosomes, the spindle, the central spindle, and the midbody flanking regions (Figure1), and interacts with both γ-tubulin and PCNT. However, loss of Kaiso does not cause obvious centrosome or mitotic defects [71,72]. CTCF is a Cells 2020, 9, 1554 7 of 27 transcriptional regulator that physically interacts with Kaiso [73]; it localizes to the centrosomes, the spindle poles and the midbody dark zone in HeLa cells (Figure1), but the mitotic e ffects of its depletion were not investigated [74]. SNAP45, a TF required for transcription of snRNA genes, localizes to the centrosomes, the spindle poles, and the midbody flanking regions (Figure1). In HeLa cells, RNAi-mediated depletion of SNAP45 leads to multiple mitotic defects, including morphologically abnormal spindles and defective chromosome segregation. These aberrant mitotic are at least in part the consequence of additional primary defects in chromosome condensation and sister chromatid pairing at metaphase [75]. Phosphorylated ELK1 localizes to the centrosomes, the spindle, the central spindle, and the midbody flanking regions in human cells [76] (Figure1). ELK1 physically interacts with Aurora A [76], and its loss causes mitotic delay but no obvious mitotic defects [20]. The Runt-related TFs (RUNX1, RUNX2, and RUNX3) are important developmental regulators with roles in carcinogenesis [77]. They also have multiple mitotic roles, but it is currently unclear whether some of these roles are direct. Human RUNX1 positively regulates transcription of genes encoding components of the spindle assembly checkpoint (SAC) machinery such as BUBR1/BUB1B, BUB1, and NEK6 [78]. During human cell mitosis, following RUNX3 phosphorylation, the RUNX proteins localize to the centrosomes and the midbody flanking regions [79] (see Figure1 for mitotic localization of individual RUNX proteins). It has been also shown that the RUNX factors co-precipitate with centrosomal proteins such as γ-tubulin and the centriolar component rootletin (CROCC) [80]. However, individual depletion of each of the RUNX proteins results in reduced cyclin B1 accumulation and delayed mitotic entry, but does not cause specific centrosome-related mitotic defects [79,80]. Several proteomic studies have shown that the centrosomes contain hundreds of different proteins, but only a fraction of them (approximately one fourth) is known to be involved in centriole structure and duplication or PCM-driven MT nucleation [81–83]. The functions of the centrosome-associated proteins that do not appear to be involved in centrosome activity, such as p53, SP1, SF-1, Kaiso, CTCF, ELK1, and RUNX TFs, are unclear. It cannot be excluded that some of these proteins play an as yet undefined direct function required for proper centrosome behavior. Alternatively, it is possible that their localization and accumulation in the centrosome helps them to interact with other proteins hosted in this subcellular structure, or to keep them sequestered until they become necessary for certain cellular processes. Recent work has provided some clues on how proteins without direct centrosome-specific functions can concentrate in this organelle. It has been suggested that centrosome assembly is facilitated by the ability of several centrosomal proteins to undergo liquid–liquid phase transition and form liquid-like droplets that concentrate cytoplasmic components and then fuse to form the PCM around the centrioles [84]. These droplets may include cytoplasmic proteins that will become spurious centrosome components. Interestingly, the centrosomes harbor several proteins involved in the DNA damage response (DDR), including the ATM, ATR, CHK1, and CHK2 kinases [85]. The ATM and ATR kinases are recruited to the sites of DNA damage and initiate a signaling cascade by phosphorylating the effector CHK1 and CHK2 kinases, ultimately leading to p53 activation [57,85]. There is evidence that DNA damage causes aberrant centriole disengagement, leading to centrosome amplification ([85] and references therein), a condition that is thought to promote tumor initiation and progression [86]. However, it remains unclear whether the centrosomal fractions of the DDR kinases and p53 have roles in DNA damage signaling and/or centrosome amplification.

2.3. TFs with Moonlighting Functions at the Midbody Many of the TFs that concentrate in the centrosomes are also enriched at the midbody. For example, ATF5, OCT1, and CTCF accumulate in the midbody dark zone, while SNAP45, ELK1, RUNX3, and Kaiso localize to regions that flank the dark zone (Table1; Figure1). The possible roles of these proteins in cytokinesis have not been thoroughly investigated. Thus, the reason for their double localization is intriguing but currently unknown. These proteins might play some direct functions in completion of cytokinesis, but it is also possible that they are hosted in the midbody without affecting cytokinesis. Cells 2020, 9, 1554 8 of 27

In this respect, it is worth noting that proteomic analysis of isolated midbodies has detected many proteins with no obvious roles in the cytokinetic process [87]. However, the β (CBFβ/CBFB) and the YAP/YAP1 transcriptional regulator, appear to play moonlighting functions in cytokinesis. In human cells, CBFβ accumulates in the flanking regions of the midbody and co-precipitates with the RUNX TFs and the regulatory light chain 3 (MRLC3/MYL12A) [88]. RNAi-mediated depletion of CBFβ or overexpression of the CBFβ-SMMHC (smooth muscle myosin heavy chain) leukemogenic fusion protein results in midbody abnormalities and mislocalization of PRC1 [88]. PRC1 cross-links antiparallel MTs in the midbody and is essential for completion of cytokinesis [89]. YAP is an interacting partner of the human RUNX proteins that accumulate in the midbody dark zone. YAP is phosphorylated during mitosis and this modification is essential for its function in cytokinesis. YAP co-precipitates with the polarity scaffold protein PATJ that is also enriched in the midbody dark zone and whose knockdown phenocopies the abscission defect elicited by YAP depletion [90]. Thus, although there is no compelling evidence for a role of the RUNX proteins in cytokinesis, the RUNX interacting partners CBFβ and YAP appear to have moonlighting functions in completion of cytokinesis.

2.4. TFs That Localize to the Spindle Many TFs associate with interphase MTs and this association is thought to regulate TF ingress into the nucleus [91]. For example, it has been reported that p53 binds to MTs and is actively transported towards the nucleus in a dynein-dependent manner [92]. Other TFs exhibit a robust association with the spindle MTs, and some of them appear to have moonlighting mitotic functions. One of these factors is (Aire), a non-canonical TF involved in the immune tolerance processes [93]. Aire associates with the metaphase spindles and ana-telophase central spindles of mouse embryonic stem (mES) cells, and physically interacts with several spindle proteins including AURKB, the centrosomal proteins CEP55 and centrobin (CNTROB), the augmin-like proteins HAUS5 and HAUS8, and the CLASP1 and CLASP2 regulators of MT dynamics [94]. Aire conditional (floxed) knockout revealed that Aire loss results in morphologically abnormal spindle poles and centrosome amplification in mES cells [94]. These results strongly suggest that Aire has a moonlighting mitotic function but the mechanisms by which Aire controls spindle assembly and centrosome number remain to be determined. Other transcriptional regulators with possible direct mitotic functions are the components of multiprotein complexes containing histone deacetylase 3 (HDAC3), which repress transcription [95]. HDAC3, the nuclear corepressor (N-CoR/NCOR1), the -β-like protein 1 (TBL1/TBL1X), and the TBL1-related protein 1 (TBLR1/TBL1XR1) all associate with the metaphase and anaphase spindles, but not with telophase spindles; RNAi-mediated depletion of either HDAC3 or N-CoR results in morphologically abnormal spindles and chromosome misalignment in HeLa cells [96]. It has been also shown that these mitotic defects are a specific consequence of disruption of HDAC3 deacetylase activity, whereas they are not elicited by treatment with the RNA polymerase II inhibitor α-amanitin [96]. Thus, even if there is no compelling evidence for a direct physical interaction of the HDAC3-associated proteins with spindle components, the extant data suggest that HDAC3 regulates mitotic spindle formation in a transcription-independent manner. Two additional TFs with roles in spindle assembly are mouse Egr3 and TFIIB, which associate with the meiotic spindles of mouse females. Egr3 localizes to the spindles, but does not appear to interact with MTs, and its function in meiotic division has not been investigated [97]. TFIIB also localizes to meiotic spindles; RNAi against TFIIB or anti-TFIIB antibody injection into the oocyte results in morphologically abnormal spindles and chromosome misalignment. However, there are no data on biochemical interactions between TFIIB and spindle components [98]. Thus, current results do not allow a conclusion on whether Egr3 and TFIIB play direct roles in meiotic spindle assembly. Cells 2020, 9, 1554 9 of 27

2.5. TFs with Roles in the Spindle Assembly Checkpoint (SAC) TFs have been also implicated in the assembly and functioning of the SAC machinery. Several TFs have been shown to regulate transcription of the SAC components. As mentioned earlier, loss of RUNX1 negatively affects transcription of the BUB1 and NEK6 SAC genes [78]. Furthermore, BRCA1 knockdown in human cancer cells downregulates transcription of multiple mitotic genes, including the BUB1, BUBR1/BUB1B, AURKA, ESPL1, and PTTG1 SAC genes [99]. Similarly, BRCA1 mutant mouse cells display decreased expression of MAD2/MAD2L1, BUB1, BUBR1, and ZW10 [54,100]. Thus, besides its moonlighting role at centrosomes, BRCA1 also controls SAC by regulating transcription of its major components. The WT1 TF and the TIF1γ/TRIM33 transcriptional intermediary factor are both functioning in the SAC-Anaphase Promoting Complex (APC/C) machinery, but while WT1 is likely to have a moonlighting function, TIF1γ does not fit all three criteria to be considered a moonlighting protein (Table1). WT1 co-localizes and directly interacts with MAD2 throughout mitosis, and has high a ffinity for the active closed MAD2 conformation in human cells. RNAi-mediated knockout of WT1 enhances turnover of the APC/C target securin (PTTG1), leading to an accelerated metaphase-to-anaphase transition and defects in chromosome segregation. Consistent with a direct mitotic role, WT1 loss does not significantly affect the expression of genes involved in SAC and APC/C regulation [101,102]. TIF1γ physically interacts with the APC/C and its CDC20 co-factor; TIF1γ knockdown in HeLa cells negatively regulates the E3 ubiquitin ligase activity of APC/C, causing inappropriate presence of cyclin A at metaphase, chromosome misalignment, and delay in anaphase onset [103,104]. Thus, WT1 and TIF1γ play opposite functions in the SAC-APC/C system, with WT1 delaying and TIF1γ accelerating metaphase-to-anaphase transition.

2.6. Mitotic and Meiotic Roles of the TFIIH Complex Components TFIIH is an evolutionarily conserved multi-subunit complex required for transcription initiation. In humans, TFIIH consists of a seven-subunit core complex formed by XPB/ERCC3, p62/GTF2H1, p52/GTF2H4, p44/GTF2H2, p34/GTF2H3, p8/GTF2H5, and XPD/ERCC2, and a three-protein subcomplex including the CDK7 kinase, (CCNH), and MAT1/MNAT1. Mutations in XPB, XPD, and p8 lead to genetic diseases such as , , and . XPD and XPB are not only involved in transcription, but also play direct functions in nucleotide excision repair (NER) [105–107]. Certain TFIIH subunits play roles in cell division. The first indication of a role of XPB in spindle formation came from studies on the haywire (hay) gene, which encodes the Drosophila ortholog of XPB [108]. A specific mutant allele of hay fails to complement mutations in the testis-specific β2-tubulin gene, which maps to a separate . In addition, males homozygous for this allele display defects in meiotic spindles, suggesting a role of hay in spindle assembly [108–110]. XPB has been also implicated in mammalian cell mitosis. XPB localizes to the centrosomes in both monkey and human cells and co-immunoprecipitates with γ-tubulin, but the functional significance of this localization has not been investigated [111]. Another TFIIH subunit with a potential mitotic role is XPD. XPD is also part of a five-protein complex independent of TFIIH that includes MMS19, MIP18/CIAO2B, CIAO1, and ANT2/SLC25A5. XPD, MMS19, and MIP18 localize to the mitotic spindle of human cells and RNAi-mediated depletion of each of these proteins results in monopolar and multipolar spindles, and in multinucleated cells [112]. Notably, fibroblasts derived from patients with certain XPD mutations show the same mitotic phenotype observed in HeLa XPD RNAi cells [112]. However, these results do not exclude the possibility that XPD deficiency might result in subtle defects in transcription leading to abnormal mitotic division [112]. The mitotic roles of the TFIIH subunits have been also analyzed in the Drosophila embryo system. Studies using fluorescently tagged TFIIH components have shown that Hay, p8, p52, and Cdk7 exhibit similar localization patterns in live early embryos. These proteins are enriched around the metaphase chromosomes and in some cases (e.g., p52) they appear to concentrate in a region corresponding to Cells 2020, 9, 1554 10 of 27 the spindle [113]. Moreover, in embryos from p8 and hay mutant mothers or Cdk7 RNAi mothers, most spindles are morphologically aberrant and show abnormal chromosome segregation [113]. Drosophila Xpd is required for proper spindle dynamics and chromosome segregation in early embryos. It has been suggested that loss of Xpd results in faulty localization of Cdk7, which is thought to cause mitotic defects [114]. In summary, while there are several indications that some TFIIH components might have direct mitotic roles in both humans and Drosophila, a solid proof is still lacking. Perhaps, the most compelling evidence for a moonlighting function of a TFIIH subunit is provided by the studies on the Drosophila hay (XPB) gene, which genetically interacts with the β2-tubulin gene and functions in meiotic spindle formation in males.

2.7. Mitotic Functions of the KANSL and NSL Complexes The nonspecific lethal (NSL) complex is a highly conserved multiprotein assembly that regulates gene transcription in both Drosophila and mammals. The human NSL complex, also called KAT8-associated nonspecific lethal (KANSL), comprises 8 proteins (KANSL1, KANSL2, KANSL3, MCRS1, PHF20, WDR5, HCF1/HCFC1, and OGT) that associate with the MOF/KAT8 acetyltransferase to regulate transcription of a specific set of genes and contribute to stem cell identity [115–117]. The Drosophila NSL complex contains at least six evolutionarily conserved Mof-associated proteins, namely, Nsl1 (KANSL1), Dgt1 (KANSL2), Rcd1 (KANSL3), Rcd5 (MCRS1), MBD-R2 (PHF20), and Wds (WDR5). The Drosophila genome also harbors the HCF1 and OGT orthologs, but it is currently unknown whether the protein products of these genes are complexed with the NSL subunits [117]. The Drosophila NSL complex acts as a major transcriptional regulator; its subunits bind to the promoters of more than 4000 housekeeping genes, although they appear to regulate transcription of only a subset of these genes [118,119]. The components of the human and Drosophila NSL complexes appear to have direct and indirect mitotic functions, providing insight into how transcription factors evolved moonlighting mitotic functions. KANSL1 and KANSL3 localize to the spindles of HeLa cells and are particularly enriched at the spindle poles. Consistent with this localization, KANSL3, and to a lesser extent KANSL1, bind the MT minus ends in vitro; MCRS1 does not bind the MT minus ends and is recruited to the spindle poles through interactions with KANSL3 and KANSL1 [120,121]. Furthermore, these three proteins physically interact with the TPX2 and MCAK/KIF2C spindle assembly factors. Depletion of each of the three proteins destabilizes the kinetochore fibers (the MT bundles that extend from the kinetochores to the spindle poles), resulting in chromosome misalignment and faulty segregation [120,121]. These data strongly suggest that KANSL1, KANSL3, and MCRS1 form a complex that associates with the spindle MT minus ends and directly controls spindle assembly and functioning (Table1). Besides being present in the KANSL complex, WDR5 also associates with the MLLN and MLLC subunits of the proteolytically cleaved mixed-lineage leukemia 1 (MLL1/KMT2A) protein, forming a complex with methyltransferase activity that catalyzes trimethylation of histone H3 lysine 4 (H3K4) and positively regulates [122,123]. The two MLL1 subunits and WDR5 localize to the centrosomes, the spindle, the central spindle, and the midbody dark zone in human cells [124–126] (Figure1). WDR5 also interacts with the MT depolymerase KIF2A, promoting its localization to the spindle poles [125], and with several midbody proteins required for cytokinesis, including PRC1, CEP55, and the centralspindlin components MKLP1/KIF23 and CYK4/RACGAP1 [124]. Consistent with its localization and biochemical interactions, RNAi-mediated depletion of WDR5 results in morphologically abnormal and elongated spindles, aberrant chromosome behavior and segregation, and failures in cytokinesis [124,125]. Collectively, these results indicate a moonlighting mitotic role for WDR5, but this role does not appear to be the same as that played by KANSL1, KANSL3, and MCRS1. Cells 2020, 9, 1554 11 of 27

The Drosophila components of the NSL complex are also implicated in cell division, but they do not appear to serve major moonlighting functions during mitosis. Genome-wide RNAi screens performed in S2 cells showed that Dgt1 (KANSL2) is required for γ-tubulin recruitment to centrosomes [17], while Rcd1 (KANSL3) and Rcd5 (MCRS1) are required for centriole duplication, and both centriole duplication and PCM recruitment at the centrosomes, respectively [18]. Another RNAi-based screen in S2 cells showed that both Rcd1 and MBD-R2 (PHF20) are required for mitotic chromosome segregation [19]. However, these studies did not determine the mitotic localization of Dgt1, Rcd1, Rcd5, and MBD-R2, nor addressed the mechanisms leading to the mitotic phenotypes elicited by depletion of these proteins. These issues have been addressed in a more recent study that analyzed in greater detail the mitotic functions of Rcd1, Rcd5, MBD-R2, and Wds (WDR5) [127]. Expression of GFP-tagged forms of these proteins in S2 cells revealed that they are all restricted to the nucleus during interphase, but relocate to specific mitotic structures during mitosis. Both Rcd1-GFP and Rcd5-GFP accumulate at the centrosomes and the midbody and are excluded from the chromosomes, but while Rcd5-GFP accumulates in the midbody dark zone, Rcd1-GFP is enriched in the central spindles and in midbody flanking regions. In contrast, MBD-R2-GFP is exclusively enriched at the mitotic chromosomes, while Wds-GFP exhibits multiple localizations, namely, the centrosomes, the kinetochores of metaphase chromosomes, the midbody dark zone, and a discrete region of a specific chromosome [127] (Figure1). Despite their different localization patterns, RNAi-mediated depletion of Rcd1, Rcd5, MBD-R2, or Wds results in identical mitotic defects, although the frequency of these defects is lower in Wds-deficient cells. Cells depleted of each of these proteins show fewer centrosomes than control cells, suggesting a defect in centriole duplication. In addition, the same cells display frequent failures in chromosome alignment and segregation. This latter phenotype has been attributed to reduction of the levels of both Cid (CENP-A) and Ndc80 [127]. Cid is a centromere-specific histone variant required for kinetochore assembly [128] and Ndc80 is the subunit of the kinetochore complex that binds to the spindle MTs [129]. Consistent with these phenotypes, transcription of many genes encoding centromere/kinetochore proteins (e.g., cid, Mis12, and Nnf1b), or involved in centriole duplication (e.g., Sas-6, Sas-4, and asl) is strongly reduced in Rcd1, Rcd5, and MBD-R2 RNAi cells, and to a lesser extent in wds RNAi cells [127]. These results suggest that the mitotic functions of Rcd1, Rcd5, MBD-R2, and Wds are mostly indirect, with these proteins working together in interphase to stimulate transcription of genes required for centrosome duplication and kinetochore assembly (Table1). In summary, the studies on the human NSL/KANSL complex revealed that four components of this complex, KANSL1, KANSL3, MCRS1, and WDR5, meet our criteria to be genuine moonlighting proteins during mitosis; namely, they localize to specific mitotic structures, they biochemically interact with proteins enriched in these structures, and their depletion leads to defects in the same structures. However, the studies on human cells did not determine whether KANSL1, KANSL3, MCRS1, or WDR5 depletion leads to reduced transcription of critical mitotic genes, and it cannot therefore be excluded that these proteins could also have some indirect transcription-related mitotic roles. Conversely, the Rcd1, Rcd5, MBD-R2, and Wds components of the Drosophila NSL complex do not fit our criteria to be regarded as moonlighting proteins with mitotic functions, and instead appear to control cell division primarily through the transcriptional regulation of mitotic genes. However, it cannot be excluded that these NSL subunits have some minor direct mitotic functions that were not detected because they are redundant or phenotypically masked by the strong defects caused by reduced transcription of critical mitotic genes (Table1). Cells 2020, 9, 1554 12 of 27

Table 1. Mitotic roles of transcription factors.

Name Mitotic Interacting Mitotic Function Loss-of-Function Mitotic Phenotype (Organism) Localization Mitotic Proteins (Role in Cancer) Regulate mitotic gene transcription. Sak1, Fkh2 (Sp) ND ND Indirect Defective septation [24]. FKHRL1/FOXO3 Regulates mitotic gene transcription. ND ND Indirect (TS) (h) Defective mitotic exit and cytokinesis [23]. Regulates mitotic gene transcription. Zas1 (Sp) Chromosomes ND Defective chromosome condensation and Indirect segregation [25]. Degrades AURKA and AURKB mRNAs. RNA processing ERG (h) ND Multipolar spindles and aberrant spindle Indirect (TP) bodies (PBs) structure [26]. Regulates transcription of multiple mitotic genes, including BUB1, BRCA1 (h) Not relevant Not relevant Indirect (TS) BUBR1/BUB1B, AURKA, ESPL1, PTTG1, ASPM, PRC1, and [99]. Regulates SAC gene transcription, BRCA1 (m) Not relevant Not relevant including MAD2, BUB1, BUBR1, and Indirect (TS) ZW10 [54,100]. Centrosome amplification and BRCA1 (h) Centrosomes γ-tubulin DIRECT (TS) fragmentation [31–37]. Morphologically abnormal spindles in TPX2, NuMa, BRCA1 (Xe) Spindle poles BRCA1-immunodepleted Xenopus egg DIRECT XRHAMM extracts [40]. Defective accumulation of PCM; Centrosomes γ-tubulin, centriolar fragmentation, multipolar ATF5 (h) (mother centriole) DIRECT PCNT spindles; unknown function at the Midbody DZ midbody [44,45]. Centrosomes γTuRC, EB1, Defective centrosome-driven MT AKNA (m) DIRECT (mother centriole) DCTN1 regrowth after MT depolymerization [49]. Centrosomes Structurally abnormal centrosomes with YB-1/YBX1 (h) γ-tubulin, PCNT DIRECT (TP) Spindle poles reduced MT nucleation ability [50,51]. OCT1/POU2F1 (h) Centrosomes PARP-1, Abnormal mitosis; unknown function at Interacts with Kinetochores DIRECT (TP) APC1 the midbody [52–55]. BRCA1 Midbody DZ Morphologically abnormal spindles in Oct1 (Xe) ND ND Oct1-immunodepleted Xenopus egg DIRECT extracts [52]. Nubbin (Dm) Central spindle ND Unknown function at the midbody [56]. P-direct ND (OCT1 ortholog) Midbody FR Centrosome amplification and p53/TP53 (h) Centrosomes ND P-direct (TS) fragmentation [60,61,65–67]. p53 (m) Centrosomes ND Centrosome amplification [58,62–64]. P-direct Centrosome amplification and decreased SP1 (h) Centrosomes ND P-direct (TP) centrosome-driven MT nucleation [68]. Centrosome amplification and decreased SP1 (m) Centrosomes ND P-direct centrosome-driven MT nucleation [68]. SF-1/NR5A1 (m) Centrosomes ND Centrosome amplification [69,70]. P-direct Centrosomes Spindle γ-tubulin, Kaiso/ZBTB33 (h) No obvious mitotic defects [71,72]. P-direct MR (TS) Central spindle PCNT Midbody FR CTCF (h) Centrosomes Mitotic phenotype not investigated Interacts with Spindle poles ND P-direct ND (TS) [73,74]. Kaiso Midbody DZ Centrosomes SNAP45/SNAPC2 Multiple mitotic defects; defective Spindle poles ND P-direct (h) chromosome condensation [75]. Midbody FR Centrosomes Spindle P-direct MR (TP, ELK1 (h) AURKA No obvious mitotic defects [20,76]. Central spindle TS) Midbody FR Cells 2020, 9, 1554 13 of 27

Table 1. Cont.

Name Mitotic Interacting Mitotic Function Loss-of-Function Mitotic Phenotype (Organism) Localization Mitotic Proteins (Role in Cancer) Regulates transcription of BUBR1, BUB1, RUNX1 (h) Not relevant Not relevant Indirect (TP) and NEK6 [78]. Centrosomes Reduced cyclin B1 accumulation and RUNX1, RUNX2, γ-tubulin, Spindle delayed mitotic entry. No specific mitotic P-direct MR (TP) RUNX3 (h) rootletin Midbody FR defects [79,80]. CBFβ/CBFB (h) Defective midbody structure and Binds RUNX Midbody FR MRLC3 cytokinesis (abscission); polyploid cells DIRECT (TS, TP) proteins [88]. YAP/YAP1 (h) Binds RUNX Midbody DZ PATJ Defective cytokinesis (abscission) [90]. DIRECT (TS, TP) proteins AURKB, CEP55, Aire (m) Spindle CNTROB, HAUS5, Abnormal spindle poles and centrosome DIRECT (AIRE in humans) Central spindle HAUS8, CLASP1, amplification [94]. CLASP2 HDAC3 (h) (in complex with Morphologically abnormal spindles and Spindle ND P-direct (TP) NCOR1, TBL1X chromosome misalignment [96]. and TBL1XR1) Meiotic spindles of Egr3 (m) ND Meiotic phenotype not investigated [97]. P-direct ND mouse females Morphologically abnormal spindles and TFIIB (m) Meiotic spindles of ND chromosomes misalignment after P-direct (GTF2B in humans) mouse females antibody injection [98]. Kinetochores Accelerated metaphase-to-anaphase WT1 (h) (colocalizes with MAD2 transition; defective chromosome DIRECT (TS, TP) MAD2) segregation [101,102]. Chromosome misalignment; delay in CDC20, TIF1γ/TRIM33 (h) ND metaphase-to-anaphase transition P-direct (TS, TP) APC/C [103,104]. XPB/ERCC3 (h) Centrosomes γ-tubulin Mitotic phenotype not investigated [111]. Direct ND (TS) (TFIIH subunit) Spindle poles Hay (Dm) Defective meiotic spindles in males; Chromosome/ (TFIIH subunit, Testis-specific abnormal mitotic spindles and defective spindle area in P-direct orthologous to β2-tubulin chromosome segregation in embryos embryos XPB) from hay mutant mothers [108–110,113]. Chromosome/ Abnormal mitotic spindles and defective p8, p52, Cdk7 (Dm) spindle area in ND chromosome segregation in embryos P-direct (TFIIH subunits) embryos from mutant or RNAi mothers [113]. XPD/ERCC2 (h) (TFIIH subunit Spindle forms another Multipolar spindles and multinucleated (with MIP18 and ND P-direct complex with cells [112]. MMS19) MIP18 and MMS19) Abnormal mitotic spindles and defective Xpd (Dm) Regulates Cdk7 ND chromosome segregation in embryos P-direct (TFIIH subunit) localization from Xpd mutant mothers [114]. KANSL1, KANSL3, Spindle poles; MCRS1 (h) KANSL1 and TPX2, Chromosome misalignment and defective DIRECT (TP) (KANSL complex KANSL3 bind MT MCAK segregation [120,121]. subunits) minus ends WDR5 (h) Centrosomes KIF2A, Morphologically abnormal and elongated (KANSL subunit Spindle PRC1, spindles, aberrant chromosome that forms another DIRECT (TP) Central spindle MKLP1, CYK4, segregation, and failures in cytokinesis complex with Midbody DZ CEP55 [124–126]. MLL1/KMT2A) Dgt1 (Dm) (NSL subunit Diffuse Diminished γ-tubulin at centrosomes; ND P-direct orthologous to (weak on spindle) long spindles [17]. KANSL2) Rcd1 (Dm) Centrosomes Defective centriole replication; frequent (NSL subunit Indirect (P-direct Central spindle ND failures in chromosome alignment and orthologous to MR) Midbody FR segregation [18,19,127]. KANSL3) Cells 2020, 9, 1554 14 of 27

Table 1. Cont.

Name Mitotic Interacting Mitotic Function Cells 2020, 9, x FOR PEER REVIEW Loss-of-Function Mitotic Phenotype 15 of 28 (Organism) Localization Mitotic Proteins (Role in Cancer) Rcd5 (Dm) Defective centriole replication; frequent (NSL subunit (NSLKinetochores subunit Centrosomes frequent failures in chromosome (P-directIndirect (P-direct ND failures in chromosome alignment and orthologous to Midbody DZ MR) orthologous to Midbody DZ alignmentsegregation and segregation [17,18,127]. [127]. MR) MCRS1) WDR5) MBD-R2 (Dm) Defective centriole replication; frequent Name: Each human(NSL subunit TF is designated either with its HUGO acronym only, or both the name used in Indirect (P-direct Chromosomes ND failures in chromosome alignment and orthologous to MR) the cited papers and the HUGO acronym. Organism: h, human; m, mouse;segregation Dm, [19 ,127 ]. PHF20) , Sp., Schizosaccharomyces pombe, Xe, Xenopus egg extracts. Mitotic localization: Wds (Dm) Centrosomes Defective centriole replication; frequent ND, not determined;(NSL subunit Midbody DZ, midbody dark zone; Midbody FR, midbody flanking regions; Indirect (P-direct Kinetochores ND failures in chromosome alignment and orthologous to MR) localization to specific midbodyMidbody regions DZ has been decided based onsegregation the examination [127]. of published WDR5) microphotographs. Interacting mitotic proteins: ND, not determined. Mitotic function: Indirect, Name: Each human TF is designated either with its HUGO acronym only, or both the name used in the cited papers controls transcriptionand the of HUGO mitotic acronym. genes; Organism:DIRECT, h,in human; addition m, mouse;to a role Dm, inDrosophila transcription melanogaster has a ,direct Sp., Schizosaccharomyces role in mitosis, pombenamely, Xe, it Xenopusis a moonlightingegg extracts. protei Mitoticn localization:that meets ND,the 3 not criteria determined; specified Midbody in the DZ, text. midbody dark zone; P-direct, putativelyMidbody direct, FR, does midbody not meet flanking the regions;3 criteria localization but (in th toe specificcited papers) midbody is regionsthought has to been have decided a based on the examination of published microphotographs. Interacting mitotic proteins: ND, not determined. Mitotic function: direct mitotic role.Indirect, P-direct controls ND, transcription localizes to of mitotica mitotic genes; structure DIRECT, but in additionits mitotic to a rolerole in has transcription not been has a direct role in investigated. P-directmitosis, MR, namely localizes it is amoonlighting to a mitotic proteinstructure that but meets its the loss 3criteria does not specified result in in the atext. detectable P-direct, putatively direct, does not meet the 3 criteria but (in the cited papers) is thought to have a direct mitotic role. P-direct ND, localizes to mitotic phenotype;a mitotic it might structure therefore but its mitotichave either role has a notminor been or investigated. a redundant P-direct mitotic MR, function. localizes to Role a mitotic in structure but its cancer was determinedloss does by not examination result in a detectable of the mitotic pertinent phenotype; literature; it might references therefore haveare not either reported: a minor orTS, a redundant mitotic tumor suppressor;function. TP, Roletumor in cancerpromoter; was determined TS, TP, can by examination function ofeither the pertinent as a tumor literature; suppressor references or are not reported: TS, tumor suppressor; TP, tumor promoter; TS, TP, can function either as a tumor suppressor or promoter, depending promoter, dependingon the on cellular the cellular context. context.

3. Mitotic Functions3. Mitotic of Splicing Functions Factors of Splicing (SFs) Factors (SFs) RNAi-based genome-wideRNAi-based genome-wide screens carried screens out carried both outin Drosophila both in Drosophila and humanand human cells cellshave have identified identified manymany different different SFs SFs that that areare requiredrequired for for mitotic mitotic division division [15– 17[15–17,19,20],19,20] (Table (Table S2). However, S2). only a few However, onlyof a thesefew of SFs these have SFs been have suffi beenciently suff characterizediciently characterized to determine to determine whether they whether have athey direct mitotic role have a direct mitoticor are insteadrole or are controlling instead controlling the splicing the of mitoticsplicing pre-mRNAs of mitotic pre-mRNAs (Table2). (Table 2).

3.1. SFs with Indirect3.1. SFs Mitotic with IndirectFunctions Mitotic Functions An evolutionarilyAn evolutionarilyconserved SF that conserved affects SFmitosis that aindirectlyffects mitosis is encoded indirectly by isthe encoded S. cerevisiae by the cef1S. cerevisiae cef1 gene. In cef1 mutants,gene. In thecef1 singlemutants, the of single the α intron-tubulin of gene the αis-tubulin not properlygene is removed, not properly resulting removed, in resulting in reduction of thereduction tubulin oflevel the and tubulin disruption level and of disruptionspindle assembly. of spindle Cells assembly. containing Cells an containingintronless an intronless α-tubulin gene αare-tubulin resistantgene to are cef1 resistant mutations, to cef1 confirmingmutations, that confirming the splicing that thedefect splicing is responsible defect is responsible for for the the phenotypephenotype [130]. An [indirect130]. An role indirect of SFs role in ofmitotic SFs in division mitotic divisionis also suggested is also suggested by studies by studies on on human human U2AF35/U2AF1.U2AF35/U2AF1. HeLa HeLacells treated cells treated with withU2AF35U2AF35 siRNAssiRNAs exhibit exhibit strong strong defects defects in inspindle spindle morphology morphology andand chromosome chromosome segregat segregation;ion; these defectsdefectshave have been been attributed attributed to to defective defective splicing splicing of the pre-mRNA of the pre-mRNAencoding encoding the CDC27 the CDC27 component component of the APCof the/C complex.APC/C complex. The localization The localization of U2AF35 of during mitosis U2AF35 duringhas mitosis not been has investigatednot been investigated [131]. [131]. SON is anotherSON SF that is another does not SF appear that does to localize not appear to mitotic to localize structures; to mitotic its structures;depletion in its HeLa depletion in HeLa cells causes failurescells causes of centrosome failures of centrosomemigration migrationto the opposite to the oppositecell poles cell during poles during prophase, prophase, and and defective defective chromosomechromosome alignment alignment and andsegregation. segregation. These These defects defects were were ascribed ascribed to inadequate to inadequate RNA RNA splicing of a splicing of a specificspecific set set of of genes, genes, including including those those encoding ɣ -tubulin-tubulin andand the the ɣTuRC TuRC component component TUBGCP2 [132]. TUBGCP2 [132].WBP11 WBP11 and and NRDE2 NRDE2 are twoare two additional additional SFs thatSFs that indirectly indirectly control control centrosome centrosome behavior. WBP11 behavior. WBP11depletion depletion in human in human cells resultscells results in the in retention the retention of multiple of multiple inintrons the TUBGCP6 in the mRNA that TUBGCP6 mRNAspecifies that another specifiesTuRC another component, ɣTuRC leading component, to defective leading centriole to duplication.defective centriole This defect is partially duplication. Thisrescued defect by is an partially intronless rescuedTUBGCP6 by an transgene,intronless TUBGCP6 supporting transgene, the idea that supporting WBP11 controlsthe centriole idea that WBP11behavior controls indirectly centriole [behavior133]. NRDE2 indirectly is a human[133]. NRDE2 SF whose is a human depletion SF resultswhose depletion in defective splicing of results in defectivethe pre-mRNA splicing of encodingthe pre-mRNA the centrosomal encoding the protein centrosomal CEP131. protein Reduction CEP131. of the Reduction CEP131 level impairs of the CEP131 recruitmentlevel impairs of recruitment proteins such of proteins as AURKA, suchγ as-tubulin, AURKA, and γ-tubulin, CEP192 toand the CEP192 centrosomes, to the and leads to centrosomes, and leads to defective chromosome segregation. During mitosis, NRDE2 is diffuse in the cytoplasm and fails to accumulate in any mitotic structure [134].

3.2. SFs That Control Cohesin Behavior In 2014, work from four different research groups led to the identification of 27 SFs that control sister chromatid cohesion in human cells [135–138]. The multi-subunit cohesin complex holds the Cells 2020, 9, 1554 15 of 27 defective chromosome segregation. During mitosis, NRDE2 is diffuse in the cytoplasm and fails to accumulate in any mitotic structure [134].

3.2. SFs That Control Cohesin Behavior In 2014, work from four different research groups led to the identification of 27 SFs that control sister chromatid cohesion in human cells [135–138]. The multi-subunit cohesin complex holds the sister chromatids together until anaphase, ensuring a faithful chromosome segregation. Cohesin associates with DNA following replication, and this association is stabilized by sororin/CDCA5 that antagonizes WAPL, a protein that is able to release cohesin from DNA, thereby allowing sister chromatid separation [139]. Seven of the 27 SFs affecting cohesin behavior have been analyzed in detail: NHP2L1/SNU13, SART1, MFAP1, CDC5L, SNW1, PRP19/PRPF19, and UBL5. RNAi-mediated depletion of each of these SFs in human cells resulted in metaphase chromosomes with parallel sister chromatids, anaphase delay, and defective chromosome segregation. Transcript analysis revealed that depletion of these factors results in intron retention in the sororin mRNA, but not in mRNAs encoding cohesin subunits. Accordingly, the SF-dependent mitotic defect was partially restored by either the expression of an intronless sororin transgene or RNAi against WAPL. These results suggest that many SFs mediate sister chromatid cohesion indirectly, predominantly by catalyzing splicing of the sororin pre-mRNA [135–138]. However, since the phenotypic rescue elicited by intronless sororin expression is often incomplete, SFs might contribute to sister chromatid cohesion in some sororin-independent way. One possibility is that SFs directly regulate cohesin association with the chromosomes, as suggested by recent work showing that many SFs physically interact with cohesin [140]. Each of the 11 known components of cohesin was tagged with a dual FLAG-SBP peptide and expressed in cultured human cells; dual affinity purification followed by revealed that the cohesin subunits copurify with a large number of SFs, including some SFs that were previously shown to be required for sister chromatid cohesion [136]. Furthermore, it has been demonstrated that in human cells, the cohesin subunit STAG2 and some of its interacting SFs (HNRNPH/HNRNPH1, EFTUD2, and SF3B1) precisely colocalize throughout the cell cycle [140]. In conclusion, while it is likely that several SFs control cohesin behavior primarily through the regulation of sororin pre-mRNA splicing, the robust physical interaction between cohesin and SFs raises the concrete possibility that some SFs contribute directly to sister chromatid cohesion.

3.3. The Indirect and Direct Mitotic Functions of the PRP19 Complex The PRP19 complex (also known as nineteen complex or NTC) is a multifunctional protein assembly that has a conserved core of four subunits (PRP19/PRPF19, CDC5L, SPF27/BCAS2, and PLRG1). Besides splicing, this complex has been implicated in several additional processes, including DNA damage response and protein ubiquitination [141,142]. The PRP19 complex is also involved in mitosis, where it appears to play both indirect and direct functions. RNAi-mediated depletion of each component of the complex in human cells impairs kinetochore-MT attachment, causing frequent failures in chromosome segregation [143]. These mitotic aberrations are attributed to defective splicing of mitotic genes such as those that encode the dynein-dynactin components DYNC1H1, DYNLRB2, and DCTN4, which are required for proper kinetochore-microtubule attachment [143]. As already mentioned, other studies showed that PRP19- and CDC5L-depleted human cells exhibit reduced cohesion between sister chromatids, and that this defect is rescued by the expression of an intronless sororin gene [136,138]. However, the same cells, in addition to the cohesion defect, display morphologically abnormal mitotic spindles. These aberrant spindles are still present in cells expressing intronless sororin and are not observed in cells depleted of cohesin subunits [138], suggesting a cohesin-independent requirement of the PRP19 complex for spindle assembly. There is an additional study that points to a splicing-independent, direct mitotic role of the PRP19 complex [42]. siRNA-mediated knockdown of SPF27 in HeLa cells leads to downregulation Cells 2020, 9, 1554 16 of 27 of the other components of the PRP19 complex and to a severe defect in end-on attachment of MTs to the kinetochores [42], consistent with the results of Mu and coauthors (2014) [143]. Moreover, an investigation of the role of the PRP19 complex in spindle assembly around sperm nuclei in Xenopus egg extracts suggests a direct mitotic function. Xenopus extracts immunodepleted of either the PRP19 or the SPF27 ortholog produce spindles with reduced MT density in the central part, suggesting a defect in the interaction between the sperm chromatin and the spindle MTs [42]. These results argue strongly for a moonlighting, splicing-independent mitotic function of the PRP19 complex because, as mentioned earlier, Xenopus egg extracts can form a spindle in the complete absence of transcription or translation of any mRNA, except cyclin B. Collectively, these results suggest a dual mitotic function for the PRP19 complex, an indirect function required for splicing of pre-mRNAs encoding sororin and some dynein-dynactin components and a direct, as yet undefined function in mitotic spindle assembly.

3.4. SFs with Roles in the SAC and in Kinetochore-MT Interactions A protein involved in pre-mRNA splicing that is also required for SAC activity in human cells is the PRP4/PRPF4 kinase. PRP4 is required for pre-mRNA splicing in S. pombe [144] and tightly associates with U4/U6 snRNP in human cells [145,146]. PRP4 accumulates on the kinetochores of HeLa cells and mediates recruitment of SAC proteins such as MPS1/IDUA, MAD1/MAD1L1, and MAD2 to kinetochores, but there is no evidence of physical interaction between PRP4 and these SAC components [147]. PRP4 depletion results in precocious anaphase onset, lagging anaphase chromosomes, and , indicating that PRP4 deficiency compromises SAC activity [147]. Thus, PRP4 does not fit all three criteria to be considered a moonlighting protein. However, in PRP4-depleted cells, the biogenesis of SAC proteins such as BUBR1/BUB1B, MAD1, MAD2, and MPS1 is not affected, suggesting a direct role of PRP4 in the SAC machinery [147]. Studies in Drosophila and human cells have recently suggested that the conserved spliceosomal components SF3A2 and PRP31 (designated as SF3A2 and PRP31/PRPF31 in humans, and Sf3A2 and Prp31 in Drosophila) play direct mitotic roles. SF3A2 is a subunit of a conserved heterotrimeric SF3A complex of U2 snRNP; PRP31 is a component of the U4/U6.U5 tri-snRNP complex that comprises at least 30 proteins [148–150]. Drosophila Sf3A2 and Prp31 associate with the spindles of S2 tissue culture cells and bind MTs in vitro [151]. The mouse ortholog of Sf3A2 was also shown to have MT binding and bundling activities [152], suggesting that Sf3A2 plays a conserved non-canonical MT-binding function. Furthermore, Sf3A2 and Prp31 physically interact with each other and directly bind the components of the Drosophila Ndc80 complex (Ndc80, Mitch/Spc25, and Nuf2). Consistent with these findings, depletion of these SFs in Drosophila cells impairs accumulation of Ndc80 at kinetochores, leading to severe defects in chromosome alignment and segregation [151]. Comparable results were obtained in HeLa cells where SF3A2 and PRP31 were found to interact physically with each other and with HEC1/NDC80. In addition, experiments with synchronized cells showed that the SF3A2-HEC1 and PRP31-HEC1 interactions are restricted to the M phase of the cell cycle. Loss of either SF in HeLa cells results in morphologically irregular spindles, defective chromosome congression at metaphase, and reduced frequency of ana-telophases [151]. Collectively, these results suggest that SF3A2 and PRP31 directly regulate interactions among kinetochores, spindle MTs, and the Ndc80 complex in both Drosophila and human cells. Further strong evidence for a direct mitotic role of Sf3A2 and Prp31 is provided by the results of antibody injections into Drosophila embryos [151]. After fertilization, Drosophila syncytial embryos undergo 13 extremely rapid nuclear divisions that predominantly rely on already spliced mRNAs and proteins packaged into the egg by the mother, although a minor transcription wave has been reported during cycles 8–13 [153,154]. These early transcripts require the Fandango subunit of the Prp19 complex for splicing, but loss of Fandango does not affect syncytial mitoses, causing nuclear defects only during the 14th cell cycle [155]. Thus, acute inactivation of a SF by antibody injection in live early embryos (cycles 8–13) is not expected to cause splicing-dependent mitotic defects, but Cells 2020, 9, 1554 17 of 27 only defects reflecting a direct mitotic role of the targeted SF. Syncytial embryos injected with either anti-Sf3A2 or anti-Prp31 antibodies shortly before nuclear envelope breakdown (NEB) display very similar mitotic phenotypes. Mitotic divisions show a decrease in spindle length relative to controls, defective chromosome alignment at metaphase, and frequent failures in chromosome segregation. This phenotype is observed as early as one minute after antibody injection, ruling out the possibility of an indirect mitotic effect of Sf3A2 and Prp31 [151]. Indeed, one minute does not appear to be sufficient for splicing and translation of a hypothetical mitotic pre-mRNA and, most importantly, it is extremely unlikely that the protein product of this pre-mRNA needs to be replenished every minute (Table2).

Table 2. Mitotic roles of splicing factors.

Name Mitotic Interacting Loss-of-Function Mitotic Mitotic Function (Organism) Localization Mitotic Proteins Phenotype (Role in Cancer) Inefficient splicing of α-tubulin pre-mRNA. Defective spindle CEF1 (Sc) ND ND assembly. Mutant phenotype Indirect rescued by an intronless α-tubulin gene [130]. Inefficient splicing of the CDC27 pre-mRNA. Defective spindle U2F35/U2AF1 (h) ND ND Indirect (TS, TP) morphology and chromosome segregation [131]. Inefficient splicing of TUBG1, TUBGCP2, TUBGCP4, and AKT1 pre-mRNAs. Impaired centrosome SON (h) ND ND Indirect (TS) separation at prophase; defective chromosome segregation and cytokinesis [132]. Inefficient splicing of TUBGCP6 pre-mRNA. Impaired centriole WBP11 (h) ND ND duplication; mutant phenotype Indirect rescued by an intronless TUBGCP6 transgene [133]. Inefficient splicing of CEP131 pre-mRNA. Impaired centrosomal Diffuse in the NRDE2 (h) ND recruitment of γ-tubulin, AURKA, Indirect (TS, TP) cytoplasm and CEP192; defective chromosome segregation [134]. 27 different human SFs, including Intron retention in the sororin PRPF8, Some mRNA. Parallel sister chromatids at Indirect, (possibly NHP2L1/SNU13, colocalize Some copurify metaphase and defective also DIRECT) (TP: SART1, MFAP1, with the with the Cohesin chromosome segregation; mutant CDC5L, SNW1, CDC5L, SNW1, Cohesin subunits phenotype partially rescued by an U2AF2) PRP19/PRPF19, complex intronless sororin transgene UBL5, SF3B1, [135–138,140]. SNRNP200, PRPF6 PRP19/PRPF19 (h), Inefficient splicing of DYNC1H1, CDC5L (h), DYNLRB2, and DCTN4 pre-mRNAs. Indirect (TP: SPF27/BCAS2 (h), Not relevant Not relevant Impaired kinetochore-MT CDC5L, BCAS2) PLRG1 (h) (PRP19 attachment, defective chromosome complex) segregation [143]. PRP19/PRPF19 (h), Defective kinetochore-MT CDC5L (h), interaction; abnormal spindles and P-direct (TP: Diffuse ND SPF27/BCAS2 (h) defective chromosome alignment in CDC5L, BCAS2) (PRP19 complex) human cells [42,138]. Cells 2020, 9, 1554 18 of 27

Table 2. Cont.

Name Mitotic Interacting Loss-of-Function Mitotic Mitotic Function (Organism) Localization Mitotic Proteins Phenotype (Role in Cancer) Prp19 (Xe), Bcas2 Defective chromatin-MT interaction; (Xe) (PRP19 ND ND morphologically abnormal spindles DIRECT complex) in Xenopus egg extracts [42]. Defective recruitment at kinetochores of the MPS1, MAD1, and MAD2 SAC proteins. PRP4/PRPF4 (h) Kinetochores ND P-direct (TP) Precocious anaphase onset; lagging anaphase chromosomes and aneuploidy [147]. Spindle Morphologically irregular spindles; SF3A2 (h), (Mouse Sf3A2 defective chromosome congression Ndc80/HEC1 DIRECT PRP31/PRPF31 (h) binds MTs at metaphase; reduced frequency of in vitro) ana-telophases [151,152]. Reduced accumulation of Ndc80 at kinetochores and severe defects in Spindle Sf3A2 (Dm), Prp31 Ndc80, Mitch, Nuf2 chromosome alignment and (Binds MTs DIRECT (Dm) (Ndc80 complex) segregation. Anti-Sf3A2 and in vitro) anti-Prp31 injections disrupt mitosis in embryos [151]. Name: Each human SF is designated either with the HUGO acronym only, or both the name used in the cited papers and the HUGO acronym. Organism: h, human; m, mouse; Dm, Drosophila melanogaster; Sc, ; Xe, Xenopus egg extracts. Mitotic localization: ND, not determined. Interacting mitotic proteins: ND, not determined. Mitotic function: Indirect, control splicing of mitotic gene pre-mRNAs; DIRECT, in addition to a role in splicing has a direct role in mitosis, namely it is a moonlighting protein that meets the 3 criteria specified in the text. P-direct, putatively direct, does not meet the 3 criteria but (in the cited papers) is thought to have a direct mitotic role. Role in cancer was determined by examination of the pertinent literature; references are not reported: TS, tumor suppressor; TP, tumor promoter; TS, TP, can function either as a tumor suppressor or a tumor promoter, depending of the cellular context.

4. Conclusions and Perspectives The studies described here implicate many transcription and splicing factors in the regulation and proper execution of mitotic division. Several of these factors do not localize to specific mitotic structures and appear to affect mitosis indirectly by controlling transcription or splicing of pre-mRNAs that encode mitotic proteins. Other factors are enriched in specific mitotic structures, but the extant data are not sufficient to establish whether they play direct mitotic functions. A third category of factors localizes to a specific mitotic structure(s), biochemically interacts with proteins enriched in same structure(s), and causes defects in this/these structure(s) upon depletion. We have assumed that the TFs and SFs with these three properties play moonlighting mitotic functions besides their canonical functions in RNA transcription or splicing. We believe that these three properties are sufficient to conclude that a TF or a SF directly functions in mitosis. However, this conclusion would be consolidated by the demonstration that depletion of the factor under study leads to mitotic defects in a model system where mitosis is both transcription- and splicing-independent. As mentioned earlier, examples of these systems are the Xenopus egg extracts immunodepleted of a specific factor [39,41,42] and live early Drosophila embryos expressing suitable fluorescent mitotic markers injected with specific antibodies. The latter system has the additional advantage of permitting the analysis of a large number of synchronous mitoses within minutes from the antibody injection [151]. The extant data indicate that the moonlighting TFs and SFs are involved in diverse mitotic processes, including (i) centriole stability, duplication, and PCM recruiting ability; (ii) centrosome- and chromatin-driven MT nucleation; (iii) spindle formation and dynamics; (iv) kinetochore-MT interaction; (v) spindle assembly checkpoint; and (vi) cytokinesis. Thus, it appears that most if not all steps of mitosis are directly regulated by some TFs and/or SFs. This raises the question of how TFs and SFs evolved direct mitotic functions. The and the biological meaning of protein moonlighting are discussed in detail in the review of Copley (2014) [4] and in the recent book by Henderson et al. Cells 2020, 9, 1554 19 of 27

(2017) [5]. Here, we only consider the simplest scenario, assuming than TFs and SFs gradually evolved so as to acquire an additional mitotic function, while keeping their canonical roles in transcription or splicing. It is likely that the TFs and SFs described here evolved a moonlighting mitotic function through the acquisition of genetic changes that allow physical interactions with mitotic proteins, specific posttranslational modifications, or both (Tables1 and2; Figure1). In this context, it is worth noting that published work (see Tables1 and2) indicates that the TFs with putative moonlighting mitotic functions outnumber the SFs with the same functions. These data raise the question of whether TFs are more prone than SFs to evolve additional mitotic functions. This is an unlikely possibility for at least two reasons. First, the global number of TFs is at least five times higher than that of SFs [21,156,157]. Second, and most importantly, a simple search of the PubMed database using “transcription factors” as query retrieved 39 times more publications than those retrieved using “splicing factors”. Thus, taking into account that RNAi-based screens yielded more SFs than TFs (Tables S1 and S2), it is conceivable that future work will identify many additional SFs with moonlighting mitotic functions. Many TFs accumulate in specific mitotic structures, but there is not compelling evidence that these accumulations are functionally relevant (Table1; Figure1). In addition, some conserved TFs appear to have species-specific mitotic localizations and functions. For example, the mitotic localizations of the human KANSL complex subunits are not identical to those of their NSL counterparts in Drosophila. In addition, while the KANSL components appear to have integral mitotic roles, their NSL orthologs are primarily involved in transcriptional regulation of specific mitotic genes [120,121,127]. However, it has been suggested that the Drosophila NSL proteins might have relatively minor direct mitotic roles that are masked by their transcription-dependent effects on cell division [127]. More in general, we propose that most proteins that accumulate in mitotic structures have either minor moonlighting functions or are evolving towards the acquisition of these functions. Finally, we would like to note that the TFs and SFs with established or possible moonlighting mitotic functions ensure the fidelity of a series of processes, whose alteration can promote carcinogenesis, namely, centrosome duplication, kinetochore-MT interaction, spindle assembly checkpoint, and cytokinesis. Many of these factors have been already implicated in cancer promotion or suppression (Tables1 and2) and their further characterization is likely to provide additional insight into their roles in tumor development. We also believe that identification of new TFs and SFs with moonlighting mitotic functions should be an important goal of future studies, as a detailed understanding of the multiple mechanisms that regulate mitotic division will be instrumental to devise new cancer therapies.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4409/9/6/1554/s1, Table S1: Transcription factors with mitotic functions identified in large-scale RNAi screens carried out in human and Drosophila tissue culture cells. Table S2: Splicing factors with mitotic functions identified in large-scale RNAi screens carried out in human and Drosophila tissue culture cells. Author Contributions: All authors contributed to the preparation of this review and to the revision of the manuscript. M.P.S. and E.N.A. performed the most extensive critical analysis of the literature. A.V.P. and M.G. wrote the manuscript and carried out the final editing. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by a grant from Associazione Italiana per la Ricerca sul Cancro (AIRC, IG 20528 to MG), by grants from the Russian Foundation for Basic Research (18-34-00688 to GAP and 18-34-00699 to JVP) and by the Fundamental Scientific Research Program of the Siberian Branch of the Russian Academy of Sciences (project 0310-2019-0005 to AVP). Conflicts of Interest: The authors declare no conflicts of interest.

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